Naturally occurring interferons are species-specific proteins produced by various cells upon induction with viruses, double-stranded RNAs, other polynucleotides, antigens, and mitogens. Interferons exhibit multiple biological activities, including antiviral, antiproliferative, immunomodulatory, and anticellular activities. Investigation of these activities has led to the identification and characterization of at least three distinct types of human interferons, which are reported to be different proteins encoded by distinct structural genes. Interferons, which are often glycoproteins, were originally classified based on their cell source and later reclassified as alpha, beta (“β”), and gamma. Interferon-beta (“IFN-β”) is produced by fibroblasts and epithelial cells. Native interferon-beta was produced by superinducing human fibroblast cultures with polyriboinosinic acid and polyribocytidylic acid and isolating and purifying the interferon(s) thus produced by chromatographic and electrophoretic techniques. The expense and difficulty of purifying interferons in this way precluded extensive clinical testing and evaluation of interferons' therapeutic value. Isolation of interferons from natural sources remains relatively difficult and expensive.
More recently, several of the human interferon genes have been cloned using recombinant DNA (“rDNA”) technology and have been expressed in E. coli (Nagola et al. (1980) Nature 284:316; Goeddel et al. (1980) Nature 287:411; Yelverton et al. (1981) Nucleic Acids Res. 9:731; Streuli et al. (1981) Proc. Natl. Acad. Sci. USA 78:2848). Proteins or polypeptides that exhibit native interferon-beta-like properties may also be produced with rDNA technology by extracting poly-A-rich 12S messenger RNA from virally induced human cells, synthesizing double-stranded cDNA using the mRNA as a template, introducing the cDNA into an appropriate cloning vector, transforming suitable microorganisms with the vector, harvesting the microorganisms, and extracting the interferon-beta therefrom. See, for example, European Patent Application Nos. 28033 (published May 6, 1981); 32134 (published Jul. 15, 1981); and 34307 (published Aug. 26, 1981), which describe various methods for the production of interferon-beta employing rDNA techniques. The expressed proteins or polypeptides from recombinant DNA clones have been purified, tested, and found to exhibit properties similar to those of native interferons. Bacterially produced interferons thus have potential therapeutic use as antiviral and antitumor agents. The production of interferons by such bacterial fermentations yields large quantities of interferon at a relatively low cost, thereby making interferon more widely available for many uses, such as clinical studies.
Interferon-beta for use in clinical studies must be of relatively high purity and substantially uncontaminated with toxic host cell constituents, cell debris, and other extraneous chemicals introduced during the extraction and purification steps. There are several methods currently available for the preparation, recovery, and purification of IFN-β.
The methods of purification and recovery of IFN-β disclosed in U.S. Pat. Nos. 4,462,940 and 5,702,699 and similar methods produce a pure form of IFN-β that tends to form aggregates in the absence of strong solubilizers, e.g., sodium dodecyl sulfate (“SDS”). In addition, such methods (1) expose the protein to high pH conditions that may adversely affect the protein's biological properties, and (2) result in compositions containing residual amounts of SDS used to solubilize the protein during purification.
Therefore, there is a need for an improved recovery and purification process in which the IFN-β is not subjected to high alkalinity, the formulation is free or virtually free of SDS, and the protein is soluble at a pH suitable for parenteral administration. It is an object of the present invention to provide a pharmaceutically acceptable sample of IFN-β that is of relatively high purity and easily refolded during the purification and recovery process.